MammalianandPlantCellCulture Module1 PlantandAnimalCellCultureMedium Handout

Animal Culture Medium Basics Medium components Buffer(s) to maintain pH Salts for osmosis and cell needs Amino acids essential and other Growth stimulants (hormones and agonists) Serum (fetal calf/bovine) Lipids including cholesterol Vitamins Food (typically glucose) Trace Minerals for metabolism/enzyme function Nucleic Acids (deoxy and NAD) Base medium Just components without additives (DMEM, RPMI) no serum or antibiotics Complete medium 5 or 10% serum Basic medium Antibiotics or antimycotics low serum or starving medium Same as complete but with no or low levels of serum (0.5%). Used to synchronize cells (quiescence) in Go of cell cycle Selecting Media (animal cells) The choice of cell culture medium is extremely important, and significantly affects the success of cell culture experiments. Different cell types have highly specific growth requirements, and the most suitable medium for each cell type must be determined experimentally. There are no set culture conditions for growing a certain cell type. A culture grown in MEM, can probably be just as easily grown in DMEM or Medium 199. In general a good place to start is MEM for adherent cells and RPMI-1640 for suspension cells. Culture Media These contain a mixture of amino acids, glucose, salts, vitamins, and other nutrients, and are available either as a powder or as a liquid from various commercial suppliers. Requires Ions; Na+, K+, Ca++, Mg++, Cl-, PO4-, HCO3Trace elements; iron, zinc, selenium Sugars such as glucose Amino Acids Culture buffers Some media includes HEPES or MOPS buffers give additional buffer strength if CO2 levels are off. Bicarbonate system is critical thus caps of culture flasks must be vented or open to allow exchange of gasses CO2 + H2O -> H2CO3 -> H+ + HCO3Serum Serum is a partially undefined material that contains growth and attachment factors, and may show considerable variation in the ability to support growth of particular cells. Fetal calf serum (FCS) is the most frequently used serum, but for some applications less expensive sera such as horse or calf serum can be used. Different serum batches should be tested to find the best one for each cell type. FCS vs FBS Fetal Calve / Bovine Serum typically used interchangeably but are different. Fetal Calf Serum taken from newborn Calves

MammalianandPlantCellCulture Module1 PlantandAnimalCellCultureMedium Handout

Fetal Bovine Serum from fetus Some variation in content of growth factors Variability in lot to lot and location to location often times tested for several viruses that my impact cells. Either can be different if mother or calve has been nursing or grazing. L-glutamine L-glutamine is an unstable amino acid that, with time, converts to a form that cannot be used by cells, and should be added to medium just before use. Provides nitrogen for NAD NADPH and nucleotides Serves (like pyruvate) as secondary energy source for metabolism Breaks down (40%) within 3 to 4 weeks, faster if in cultured cells. In culture, glutamine breakdown generates ammonium Some supplements (glutamax) are more stable and can replace glutamine for long term culturing of slow cells Antibiotic & Antimycotic Antibiotics and fungicides can be used as a supplement to aseptic technique to prevent microbial contamination. Media & Components Media, serum, and supplements should be tested for sterility before use by incubation of a small aliquot at 37C for 48 hours. If microbial growth has occurred after this incubation, the medium or supplement should be discarded. Making Media see online video and protocols From powder or liquid form Very recipe dependent. Plant Culture Medium Plant Culture Medium Requirements Varies: Plant cell type (woody, fern, orchid) Maintenance of callus or shoot formation (stage II) Stimulation of Root and de-differentiation Protoplast, suspension or batch cultures General Components: Macronutrients, micronutrients, vitamins, amino acids, nitrogen, phosphorous, sugar, organic supplements and solidifying agents/support systems AND growth regulators (hormones) Macronutrients (macroelements) - Needed in media in large amounts and make up ~0.1% of dry weight of plant: Nitrogen supplied in form of ammonium ion (H4NO3+) and nitrate (KNO3) best if both are present and together act to buffer pH. Some amino acids can supplement N requirements or take place as the N is removed via TCA and transglutaminases High ammonium causes a pale, glassy culture (vitrification) Potassium come as counter ion with NO3-and PO4-2 Phosphorus K2HPO4, H4NO3(HPO4)2, High concentrations of phosphate will lead to ppt with Ca+2 and other cations Micronutrients (Microelements): Trace amount elements and salts necessary for growth: Fe (FeSO4) Chelated to EDTA is most critical. The complex allows for a slow continuous release and avoids free metal generation of radical oxides after reaction with water. Others include: Zn, Cu, B, and Mo. Carbon and Energy Source cultures do little if any photosynthesis (heterotrophs). Must supply carbon to metabolize ATP and other energy molecules. Sucrose is usually used Galactose, sorbitol and maltose also are used. Plant Culture Medium Organic Supplements Wide range of various needs

MammalianandPlantCellCulture Module1 PlantandAnimalCellCultureMedium Handout

Amino Acids can provide nitrogen and support for metabolism as well as biosynthesis for new proteins, lipids and nucleotides Casine (milk protein) hydrolysates typically are the source of amino acids Vitamins: Vitamin B1 (thiamin) and Vitamin B6 (nicotinic acid pyridoxine), and myo-inositol. The latter is not a vitamin but used as one for plant culture media. Activated Charcoal (AC) Used for its ability to bind hydrophobic compounds which inhibit growth. The actual role isnt always clear nor is it always included in medium. Gelling Agents (support systems) Solidified surface typically from the complex carbohydrates (nondigestible) extracted from seaweed (agar). Lots of variation between batches and suppliers Gums from plants, agarose can also be used Growth Regulators- Five main classes; auxin, cytokinin, gibberellins, abscisic acid and ethylene. Auxins- Promote cell division and growth most auxins are synthetic and not found in plants. Naturally produced 1Hindol-3-acetic acid, is unstable to both heat and light. Cytokinins Promote cell division and are produce in young leaves fruits and seeds. Ratio of Auxin and cytokinin control root formation Root initiation occurs when more auxin than cytokinin is in media and adventitious and shoot growth takes place when more cytokinin than auxin ratio Gibberellins & Abciscic acid- Regulate cell elongation and determine plant height. Gibberellins increase growth of low-density cultures, enhance callus growth and elongate dwarf plants Abscisic acid alters callus growth, enhance bud and shoot formation, and inhibit cell division. Commonly used in somatic embryogenesis Ethylene- volatile gas produced during ripening, stress, mechanical damage or infection. Produced from methyl group of methionine Nearly all plant tissues can produce Natural role is to encourage fruit ripening and flower blooming Used commercially to initiate flowering and ripen tomatoes, citrus and bananas why brown bags? Specific protein receptors for ethylene have been found which act as transcription factors Can be a problem in culture without proper air circulation